Difference between revisions of "Part:BBa K2043001"
Line 1: | Line 1: | ||
− | + | NOTOC__ | |
− | + | ||
<partinfo>BBa_K2043001 short</partinfo> | <partinfo>BBa_K2043001 short</partinfo> | ||
− | This | + | This part corresponds to <b>Catechol-1,2-dioxygenase</b> cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzymes originally comes from <i>Acinetobacter pittii</i>, which we <b>codon optimised for <i>E. coli</i></b>.<br> |
+ | In order to facilitate working with this enzyme, we added a <b>His-tag</b> at the <b>C-terminal</b>. This tag allows for purification in an easier way.<br><br> | ||
+ | <br> | ||
+ | We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. Our project consisted in the degradation of wine strains, and therefore enzymes with the ability to degrade polyphenolic molecules were of interest to us. <br> | ||
+ | In particular, Catechol-dioxygenases are good candidates because they degrade Catechol, which is structurally similar to Anthocyanins.<br> | ||
+ | |||
+ | <b>Testing the part</b><br><br> | ||
+ | |||
+ | We tested the activity of CatA using cell extract of cells expressing our protein. <br> | ||
+ | First, we performed an SDS-PAGE to check whether the protein was being expressed. <br> | ||
+ | https://static.igem.org/mediawiki/parts/1/1e/Paris_Bettencourt_notebook_GELS.jpg | ||
+ | <br><br> | ||
+ | |||
+ | The enzyme was successfully expressed, and therefore we continued to the next step, which was testing our protein's activity. <br> | ||
+ | We tested our cell extract for CatA activity in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed. <br> | ||
+ | Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product. <br> | ||
+ | https://static.igem.org/mediawiki/parts/6/63/Paris_Bettencourt_notebook_catA_good.jpg | ||
+ | |||
− | |||
− | |||
<!-- --> | <!-- --> | ||
Line 19: | Line 33: | ||
<!-- --> | <!-- --> | ||
− | <h1> | + | |
− | < | + | <h1>GFP-FBD2</h1> |
+ | |||
+ | <pre> | ||
+ | ........ | ||
+ | ;::;;::;, | ||
+ | ;::;;::;;, | ||
+ | ;;:::;;::;;, | ||
+ | .vnmmnv%vnmnv%,.;;;:::;;::;;, .,vnmnv%vnmnv, | ||
+ | vnmmmnv%vnmmmnv%vnmmnv%;;;;;;;%nmmmnv%vnmmnv%vnmmnv | ||
+ | vnmmnv%vnmmmmmnv%vnmmmmmnv%;:;%nmmmmmmnv%vnmmmnv%vnmmmnv | ||
+ | vnmmnv%vnmmmmmnv%vnmmmmmmmmnv%vnmmmmmmmmnv%vnmmmnv%vnmmmnv | ||
+ | vnmmnv%vnmmmmmnv%vnmmmmmmmmnv%vnmmmmmmmmmmnv%vnmmmnv%vnmmmnv | ||
+ | vnmmnv%vnmmmmmnv%vnmm;mmmmmmnv%vnmmmmmmmm;mmnv%vnmmmnv%vnmmmnv, | ||
+ | vnmmnv%vnmmmmmnv%vnmm;' mmmmmnv%vnmmmmmmm;' mmnv%vnmmmnv%vnmmmnv | ||
+ | vnmmnv%vnmmmmmnv%vn;; mmmmnv%vnmmmmmm;; nv%vnmmmmnv%vnmmmnv | ||
+ | vnmmnv%vnmmmmmmnv%v;; mmmnv%vnmmmmm;; v%vnmmmmmnv%vnmmmnv | ||
+ | vnmmnv%vnmmmmmmnv%vnmmmmmmmmm;; mmmmmmmmmnv%vnmmmmmmnv%vnmmmnv | ||
+ | vnmmnv%vnmmmmmmnv%vnmmmmmmmmmm;; mmmmmmmmmmnv%vnmmmmmmnv%vnmmmnv | ||
+ | vnmmnv%vnmmmmm nv%vnmmmmmmmmmmnv;, mmmmmmmmmmmmnv%vn;mmmmmnv%vnmmmnv | ||
+ | vnmmnv%vnmmmmm nv%vnmmmmmmmmmnv%;nmmmmmmmmmmmnv%vn; mmmmmnv%vnmmmnv | ||
+ | `vnmmnv%vnmmmm, v%vnmmmmmmmmmmnv%vnmmmmmmmmmmnv%v; mmmmnv%vnnmmnv' | ||
+ | vnmmnv%vnmmmm;, %vnmmmmmmmmmnv%vnmmmmmmmmmnv%;' mmmnv%vnmmmmnv | ||
+ | vnmmnv%vnmmmm;;, nmmm;' mmmm;;' mmmnv%vnmmmmnv' | ||
+ | `vnmmnv%vnmmmmm;;,. mmnv%v;, mmmmnv%vnmmmmnv' | ||
+ | `vnmmnv%vnmmmmmmnv%vnmmmmmmmmnv%vnmmmmmmnv%vnmmmmmnv%vnmmmmnv' | ||
+ | `vnmvn%vnmmmmmmnv%vnmmmmmmmnv%vnmmmmmnv%vnmmmmmnv%vnmmmnv' | ||
+ | `vn%vnmmmmmmn%:%vnmnmmmmnv%vnmmmnv%:%vnmmnv%vnmnv' | ||
+ | |||
+ | </pre> |
Revision as of 17:09, 21 October 2016
NOTOC__ catA from Acinetobacter pittii, codon optimized for E. coli
This part corresponds to Catechol-1,2-dioxygenase cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzymes originally comes from Acinetobacter pittii, which we codon optimised for E. coli.
In order to facilitate working with this enzyme, we added a His-tag at the C-terminal. This tag allows for purification in an easier way.
We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. Our project consisted in the degradation of wine strains, and therefore enzymes with the ability to degrade polyphenolic molecules were of interest to us.
In particular, Catechol-dioxygenases are good candidates because they degrade Catechol, which is structurally similar to Anthocyanins.
Testing the part
We tested the activity of CatA using cell extract of cells expressing our protein.
First, we performed an SDS-PAGE to check whether the protein was being expressed.
The enzyme was successfully expressed, and therefore we continued to the next step, which was testing our protein's activity.
We tested our cell extract for CatA activity in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed.
Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
GFP-FBD2
........ ;::;;::;, ;::;;::;;, ;;:::;;::;;, .vnmmnv%vnmnv%,.;;;:::;;::;;, .,vnmnv%vnmnv, vnmmmnv%vnmmmnv%vnmmnv%;;;;;;;%nmmmnv%vnmmnv%vnmmnv vnmmnv%vnmmmmmnv%vnmmmmmnv%;:;%nmmmmmmnv%vnmmmnv%vnmmmnv vnmmnv%vnmmmmmnv%vnmmmmmmmmnv%vnmmmmmmmmnv%vnmmmnv%vnmmmnv vnmmnv%vnmmmmmnv%vnmmmmmmmmnv%vnmmmmmmmmmmnv%vnmmmnv%vnmmmnv vnmmnv%vnmmmmmnv%vnmm;mmmmmmnv%vnmmmmmmmm;mmnv%vnmmmnv%vnmmmnv, vnmmnv%vnmmmmmnv%vnmm;' mmmmmnv%vnmmmmmmm;' mmnv%vnmmmnv%vnmmmnv vnmmnv%vnmmmmmnv%vn;; mmmmnv%vnmmmmmm;; nv%vnmmmmnv%vnmmmnv vnmmnv%vnmmmmmmnv%v;; mmmnv%vnmmmmm;; v%vnmmmmmnv%vnmmmnv vnmmnv%vnmmmmmmnv%vnmmmmmmmmm;; mmmmmmmmmnv%vnmmmmmmnv%vnmmmnv vnmmnv%vnmmmmmmnv%vnmmmmmmmmmm;; mmmmmmmmmmnv%vnmmmmmmnv%vnmmmnv vnmmnv%vnmmmmm nv%vnmmmmmmmmmmnv;, mmmmmmmmmmmmnv%vn;mmmmmnv%vnmmmnv vnmmnv%vnmmmmm nv%vnmmmmmmmmmnv%;nmmmmmmmmmmmnv%vn; mmmmmnv%vnmmmnv `vnmmnv%vnmmmm, v%vnmmmmmmmmmmnv%vnmmmmmmmmmmnv%v; mmmmnv%vnnmmnv' vnmmnv%vnmmmm;, %vnmmmmmmmmmnv%vnmmmmmmmmmnv%;' mmmnv%vnmmmmnv vnmmnv%vnmmmm;;, nmmm;' mmmm;;' mmmnv%vnmmmmnv' `vnmmnv%vnmmmmm;;,. mmnv%v;, mmmmnv%vnmmmmnv' `vnmmnv%vnmmmmmmnv%vnmmmmmmmmnv%vnmmmmmmnv%vnmmmmmnv%vnmmmmnv' `vnmvn%vnmmmmmmnv%vnmmmmmmmnv%vnmmmmmnv%vnmmmmmnv%vnmmmnv' `vn%vnmmmmmmn%:%vnmnmmmmnv%vnmmmnv%:%vnmmnv%vnmnv'